Separating with an agent comprising an aliphatic ester of a polysaccharide

ABSTRACT

A separation agent essentially comprises an aliphatic or aromatic ester of a polysaccharide, except for cellulose acetate and an aromatic ester of cellulose. It is useful for separation of various chemical substances, especially optical resolution of optical isomers.

This is a division of Ser. No. 07/640,079, filed Jan. 11, 1991, now U.S.Pat. No. 5,089,138, which is a division of Ser. No. 07/430,736, filedNov. 2, 1989, now U.S. Pat. No. 5,041,226, which is a division of Ser.No. 07/225,066, filed Jul. 27, 1988, now U.S. Pat. No. 4,892,659, whichis a division of Ser. No. 07/018,814, filed Feb. 18, 1987, now U.S. Pat.No. 4,786,415, which is a continuation of Ser. No. 06/716,790, filedMar. 27, 1985, now abandoned.

The invention relates to a separation agent which essentially comprisesan aliphatic ester of a polysaccharide, excluding for cellulose acetate.It is useful for separation of various chemical substances, especiallyoptical resolution of optical isomers.

The resolving agent of the present invention can be used for separationof all sorts of chemical substances, particularly for optical resolutionof them.

It has been well known that optical isomers of a chemical compound haveeffects different from each other in vivo generally. Therefore, it isimportant to obtain optically pure compounds for the purposes ofimproving medicinal effects per unit dose of them and removing adversereactions thereof and damage from them in medical, agricultural andbiochemical fields. A mixture of optical isomers has been opticallyresolved by preferential crystallization or diastereomer process.However, varieties of compounds capable of being optically resolved bythese processes are limited and these processes require a long time anda much labor. Under these circumstances, development of a technique ofconducting the optical resolution by an easy chromatographic process haseagerly been demanded.

Chromatographic optical resolution has been investigated from old times.However, resolving agents developed heretofore have problems that theyhave only an unsatisfactory resolution efficiency, compounds to beresolved should have a specific functional group and their stability isonly poor. Thus, it has been difficult to optically resolve all sorts ofcompounds with satisfactory results.

An object of the present invention is to provide a resolving agenthaving a chemical structure different from those of known resolvingagents, particularly the resolving agents containing cellulosetriacetate, as an effective component, and therefore, the resolvingcharacteristics different from those of the known ones or a higherfaculty of discriminating and identifying the optical isomers.

The above-mentioned object of the present invention is attained by aresolving agent containing an aliphatic acid ester of a polysaccharide,excluding cellulose acetate, as effective component.

The resolving agent of the invention exhibits preferably differentpowers of adsorbing different optical isomers of a given compound.

The term "polysaccharides" herein involves any optically activepolysaccharide selected from the group consisting of synthetic, naturaland modified natural polysaccharides. Among them, those having highlyregular linkages are preferred. Examples of them include β-1,4-glucans(celluloses), α-1,4-glucans (amylose and amylopectin), α-1,6-glucan(dextran), β-1,6-glucan (pustulan), β-1,3-glucans such as curdlan andschizophyllan, α-1,3-glucan, β-1,2-glucan (Crown gall polysaccharide),β-1,4-galactan, β-1,4-mannan, α-1,6-mannan, β-1,2-fructan (inulin),β-2,6-fructan (levan), β-1,4-xylan, β-1,3-xylan, β-1,4-chitosan,β-1,4-N-acetylchitosan (chitin), pullulan, agarose and alginic acid.Still preferred ones are those capable of easily yielding highly purepolysaccharides, such as cellulose, amylose, β-1,4-chitosan, chitin,β-1,4-mannan, β-1,4-xylan, inulin, curdlan and α-1,3-glucan.

These polysaccharides have a number-average degree of polymerization(average number of pyranose or furanose rings in the molecule) of atleast 5, preferably at least 10. Though there is provided no upper limitof the degree of polymerization, it is preferably 500 or less from theviewpoint of the care of handling.

The term "aliphatic esters of polysaccharides" herein refers topolysaccharides wherein 30 to 100%, preferably 85 to 100%, on average ofthe total hydroxyl groups are esterified with an aliphatic carboxylicacid. The "aliphatic carboxylic acid" has an acyclic or cyclichydrocarbon skeleton having 1 to 20 carbon atoms, being free of anyaromatic or heteroaromatic group. Examples of them include acetic acid(which is excluded when the polysaccharide is cellulose), propionic acidand cycloalkanecarboxylic acids having a 3-, 4-, 5-, 6- or 7-memberedring. They include also those aliphatic carboxylic acids having anunsaturated bond, such as propiolic and acrylic acid derivatives. Thesecarboxylic acids may have a substituent containing a hetero atom such asa halogen, nitrogen or oxygen.

In the polysaccharide, the hydroxyl groups other than those forming thelinkage bond with the above-mentioned carboxylic acid may be present inthe form of free hydroxyl groups or they may be esterified, etherifiedor carbamoylated so far as the resolving ability of the resolving agentis not damaged.

The esterification for forming the fatty acid esters of thepolysaccharides used in the present invention may be conducted by aknown process for the esterification of cellulose or amylose (see, forexample, "Dai-Yuki Kagaku" 19, `Tennen Kobunshi Kagaku I` published byAsakura Book Store, p. 124). For example, generally used esterifyingagents are anhydrides and halides of corresponding carboxylic acids,particularly acid chlorides. It is preferred to use a tertiary aminebase or Lewis acid as the catalyst. The reaction solvent may be anysolvent so far as it does not inhibit the esterification reaction, suchas pyridine or quinoline which acts also as the base. Frequently, acatalyst such as 4-(N,N-dimethylamino)pyridine is effective inaccelerating the reaction.

Further, a corresponding carboxylic acid combined with a dehydratingagent may also be reacted with the polysaccharide to obtain the ester.

Since most of the polysaccharides used as the starting material have alow reactivity, it is preferable that, they are activated bydissolution/reprecipitation or dissolution/freeze drying treatment or byusing a reaction solvent in which the polysaccharides are soluble.

The resolving agent of the present invention is used for the purpose ofresolving compounds and optical isomers thereof generally according to achromatographic method such as gas, liquid or thin layer chromatographicmethod. Further, the resolving agent may be used in membrane resolutionmethod.

In using the resolving agent of the present invention in the liquidchromatography, there may be employed a method wherein the powderedresolving agent is packed in a column, a method wherein a capillarycolumn is coated with the resolving agent, a method wherein a capillaryis made from the resolving agent to use the inner wall thereof and amethod wherein the resolving agent is spun and bundled up to form acolumn. Among them, the method wherein the powdered resolving agent isemployed is most general.

The separation agent of the invention is preferably used in the form ofpowder. It is obtained by crushing the agent or forming it intospherical beads. The particle size which varies depending on the size ofa column or plate used is 1 μm to 10 mm, preferably 1 to 300 μm. Theparticles are preferably porous.

It is preferred to support the resolving agent on a carrier so as toimprove the resistance thereof to pressure or to prevent swelling orshrinkage thereof due to solvent exchange and from the viewpoint of thenumber of theoretical plates. The suitable size of the carrier whichvaries depending on the size of the column or plate used is generally 1μm to 10 mm, preferably 1 to 300 μm. The carrier is preferably porousand has an average pore diameter of 10 Å to 100 μm, preferably 50 to50,000 Å. The amount of the resolving agent to be supported is 1 to 100wt. %, preferably 5 to 50 wt. %, based on the carrier.

The resolving agent may be supported on the carrier by either chemicalor physical means. The physical means include one wherein the resolvingagent is dissolved in a suitable solvent, the resulting solution ismixed with a carrier homogeneously and the solvent is distilled off bymeans of a gaseous stream under reduced pressure or heating and onewherein the resolving agent is dissolved in a suitable solvent, theresulting solution is mixed homogeneously with a carrier and the mixtureis dispersed in a liquid incompatible with said solvent by stirring todiffuse the solvent. The resolving agent thus supported on the carriermay be crystallized, if necessary, by heat treatment or the like.Further, the state of the supported resolving agent and accordingly itsresolving power can be modified by adding a small amount of a solventthereto to temporarily swell or dissolve it and then distilling thesolvent off.

Both porous organic and inorganic carriers may be used, though thelatter is preferred. The suitable porous organic carriers are thosecomprising a high molecular substance such as polystyrene,polyacrylamide or polyacrylate. The suitable porous inorganic carriersare synthetic or natural products such as silica, alumina, magnesia,titanium oxide, glass, silicate or kaolin. They may be surfacetreated soas to improve their affinity for the resolving agent. The surfacetreatment may be effected with an organosilane compound or by plasmapolymerization.

In using the resolving agent of the present invention in the resolutionof compounds or optical isomers, the resolving characteristics thereofmay vary sometimes depending on physical properties thereof such asmolecular weight, crystallinity and orientation, even though they arechemically similar. Therefore, the resolving characteristics of theresolving agent may be altered according to the use thereof by suitablyselecting the solvent used in that step or by physical treatments suchas heat treatment, etching or swelling with the liquid after said stepin any of the above-mentioned processes.

In liquid or thin layer chromatography, any developer may be used exceptthose in which the resolving agent is soluble or which are reactive withthe resolving agent. In case the resolving agent has been bound to thecarrier by the chemical process or it has been insolubilized bycrosslinking, any solvent other than a reactive liquid may be used. As amatter of course, it is preferred to select the developer afterexamination of various developers, since the resolving characteristicsof chemical substances or optical isomers vary depending on thedeveloper used.

In the thin layer chromatography, a layer having a thickness of 0.1 to100 mm and comprising the resolving agent in the form of particles ofabout 0.1 μm to 0.1 mm-and, if necessary, a small amount of a binder isformed on a supporting plate.

In the membrane resolution process, the resolving agent is used in theform of a hollow filament or film.

The resolving agent of the present invention containing the aliphaticester of the polysaccharide as the effective component is effective forthe resolution of various compounds. Particularly, it is quite effectivefor the resolution of optical isomers which are quite difficult toresolve. Either one of the optical isomers to be resolved is selectivelyadsorbed on the resolving agent.

The resolving agents of the present invention were compared withcellulose triacetate after supporting them on silica beads. In theresolution of benzoin, a separation factor α of 1.10 was obtained withcellulose triscyclopropanecarboxylate, while the value of α obtainedwith cellulose triacetate was 1.05. In the resolution of transstilbeneoxide, the values α obtained with mannan triacetate and cellulosetriacetate were 1.51 and 1.22, respectively. Thus, it will be understoodthat the resolving agents of the present invention have excellenteffects.

The separation agent according to the invention is useful for opticalresolution as shown above. In addition, it serves for separation ofgeometrical isomers and polymers having different molecular weightranges from each other. They have not easily been separated in the stateof prior arts.

The following examples will further illustrate the present invention,which by no means limit the invention. In the examples, the terms aredefined as follows: ##EQU1##

SYNTHESIS EXAMPLE 1

10 g of silica beads (LiChrospher SI 1000; a product of Merck & Co.) wasplaced in a 200 ml round-bottom flask with a side arm. Aftervacuum-drying in an oil bath at 120° C. for 3 h, N₂ was introducedtherein. 100 ml of toluene which had been preliminarily distilled in thepresence of CaH₂ was added to the silica beads. 3 ml ofdiphenyldimethoxysilane (KBM 202; a product of Shin'etsu Kagaku Co.,Ltd.) was added to the mixture and they were stirred together and thenreacted at 120° C. for 1 h. After distilling off 3 to 5 ml of toluene,the reaction was carried out at 120° C. for 2 h. The mixture wasfiltered through a glass filter, washed with 50 ml of toluene threetimes and then with 50 ml of methanol three times and dried in vacuum at40° C. for 1 h.

About 10 g of the silica beads were placed in the 200 ml round-bottomflask with a side arm. After vacuum drying at 100° C. for 3 h, thepressure was returned to the atmospheric pressure and the mixture wascooled to room temperature. Then N₂ was introduced therein. 100 ml ofdistilled toluene was added to the dried silica beads. 1 ml ofN,O-bis(trimethylsilyl)acetamide (a trimethylsilylating agent) was addedthereto and the mixture was stirred to effect the reaction at 115° C.for 3 h. The reaction mixture was collected with a glass filter, washedwith toluene and dried under vacuum for about 4 .

SYNTHESIS EXAMPLE 2

Cellulose triacetate (a product of Daicel Ltd.) having a number-averagedegree of polymerization of 110 and a degree of substitution of 2.97 wasdissolved in 1 l of acetic acid (a product of Kanto Kagaku Co.). 5.2 mlof water and 5 of conc. sulfuric acid were added to the resultingsolution and the reaction was carried out at 80° C. for 3 h. Thereaction mixture was cooled and sulfuric acid was neutralized with anexcess amount of an aqueous magnesium acetate solution. The resultingsolution was added to 3 l of water to precipitate cellulose triacetatehaving a reduced molecular weight. After collection with a glass filter(G3), it was dispersed in 1 liter of water. After collection followed byvacuum drying, the obtained product was dissolved in methylene chlorideand reprecipitated from 2-propanol. The dissolution and thereprecipitation were repeated twice to effect the purification. Theproduct was dried. According to the IR and NMR spectra, the product wasidentified with cellulose triacetate. The number-average molecularweight of the product as determined by vapor pressure osmometry was7900, which corresponded to the number-average degree of polymerizationof 27. The vapor pressure osmometry was conducted with a vapor pressureosmometer Corona 117 using a solvent mixture of chloroform/1% ethanol.

60 g of the obtained cellulose triacetate was dispersed in 200 ml of2-propanol. 60 ml of 100% hydrazine hydrate (a product of NakraiChemicals Ltd.) was added dropwise slowly to the dispersion under gentlestirring. The suspension was maintained at 60° C. for 3 h and theresulting cellulose was filtered through a glass filter, washed withacetone repeatedly and dried in vacuo at 60° C. In the IR spectrum ofthe product, no absorption due to the carbonyl group at around 1720 cm⁻¹was observed and the IR spectrum coincided with that of cellulose.

SYNTHESIS EXAMPLE 3 Synthesis of Cellulose Triscyclopropanecarboxylate

90 ml of dehydrated pyridine, 15.4 ml of dehydrated triethylamine and100 ml of 4-dimethylaminopyridine were added to 3 g of the celluloseobtained in Synthesis Example 2. 17.4 g of cyclopropanecarbonyl chloridewas added thereto under stirring and the mixture was stirred at 100° C.for 5 h to carry out the reaction. After cooling, the product was addedto 500 ml of ethanol under stirring to form a precipitate, which wascollected with a glass filter and washed thoroughly with ethanol. Afterdrying in vacuo, the product was dissolved in 30 ml of methylenechloride. After an insoluble matter was removed, the residue wasreprecipitated from 400 ml of ethanol. The precipitate was collected,washed with ethanol, dehydrated and dried to obtain 4.2 g of cellulosetriscyclopropanecarboxylate. The product was dissolved in methylenechloride and the solution was applied to a sodium chloride plate anddried. The infrared absorption spectrum of the product had the followingcharacteristic absorption bands:

2900 to 3100 cm⁻¹ : stretching vibration of C--H of cyclopropane ring,

1740 cm⁻¹ : stretching vibration of C═O of carboxylic acid ester,

1450 cm⁻¹ : deformation vibration of C--H of cyclopropane ring,

1260 cm⁻¹ : stretching vibration of C--O of ester, and

1060 to 1160 cm⁻¹ : stretching vibration of C--O--C of cellulose.

Substantially no absorption at around 3450 cm⁻¹ due to OH of cellulosewas observed. This fact suggested that the product substantiallycomprised a trisubstituted compound. In the proton NMR spectrumdetermined in CDCl₃, the characteristic absorption bands were asfollows:

0.6 to 1.2 ppm: methylene proton of cyclopropane ring,

1.4 to 1.9 ppm: methyne proton of cyclopropane ring, and

3.4 to 5.4 ppm: protons of the cellulose ring and methylene in position6

The ratio of these absorption intensities was 12:3:7, which coincidedwith that of the trisubstituted compound.

EXAMPLE 1

1.2 g of cellulose triscyclopropanecarboxylate obtained in SynthesisExample 3 was dissolved in 10 ml of dichloromethane. 3.2 g of the silicabeads obtained in Synthesis Example 1 were impregnated with 7.5 ml ofthe solution. The solvent was distilled off under reduced pressure toobtain a powdery, supported material.

EXAMPLE 2

Albumens of seeds of ivory palm were treated by a process disclosed inliterature [see G. O. Aspinall et al., "J. Chem. Soc.", 3184 (1953)] toobtain mannan B from a high molecular weight fraction. 1.5 g of powderof mannan B was mixed with 50 ml of dehydrated pyridine, 10 ml ofdehydrated triethylamine and 100 mg of 4-dimethylaminopyridine. 20 ml ofacetic anhydride was added thereto under stirring. The reaction wascarried out at 100° C. for 6.5 h. After cooling, the product was addedto 400 ml of ethanol under stirring to form a precipitate, which wasfiltered and collected with a glass filter, washed thoroughly withethanol and dried in vacuo to obtain 1.7 g of a product. The product,i.e. β-1.4-mannan acetate, was insoluble in methylene chloride. In theIR absorption spectrum, a considerably intense absorption due to OH wasobserved at around 3450 cm⁻¹. The product was acetylated again asfollows: the whole of the product was dissolved in 10 ml ofdichloromethane, 1.5 ml of acetic anhydride and 2.5 ml oftrifluoroacetic anhydride. The solution was kept at room temperature forone day. A volatile matter was distilled off under reduced pressure andthe residue was washed with ethanol. The product was dried. 1.2 g of theproduct was dissolved in 4 ml of dichloromethane, 3 ml oftrifluoroacetic acid and 0.5 ml of acetic anhydride. The solution waskept at room temperature for a whole day and night. 3.2 g of the silicabeads obtained in Synthesis Example 1 were impregnated with 7.5 ml ofthe solution. The solvent was distilled off under reduced pressure toform a powdery, supported material. In the I.R. absorption spectrum ofthe product recovered from a part of said solution by precipitation, anabsorption peak peculiar to the acetate was observed but the absorptiondue to the free hydroxyl group (at around 3500 cm⁻¹) was quite weak tosuggest that the product was a trisubstituted compound.

APPLICATION EXAMPLE 1

The silica beads carrying cellulose triscyclopropanecarboxylate obtainedin Example 1 were packed in a stainless steel column having a length of25 cm and an inner diameter of 0.46 cm by slurry process. The highperformance liquid chromatograph used was TRIROTAR-SR (a product ofJapan Spectroscopic Co., Ltd.) and the detector was UVIDEC-V. Theresults of the resolution of racemic compounds of trans-stilbene oxideare shown in Table 1. of trans-stilbene oxide are shown in Table 1.

                                      TABLE 1                                     __________________________________________________________________________                    capacity                                                                            Resolution                                                                          Rate of                                                           ratio factor                                                                              separation                                                                          Flow rate                                   racemates       k.sub.1 '                                                                        k.sub.2 '                                                                        α                                                                             Rs    ml/min                                      __________________________________________________________________________     ##STR1##       0.84                                                                             1.0                                                                              1.20  0.5   1.0                                          ##STR2##       4.64                                                                             5.12                                                                             1.10  0.84  1.0                                         __________________________________________________________________________     Solvent: hexane/2propanol (9:1)                                          

APPLICATION EXAMPLE 2

The silica beads carrying β-1,4-mannan triacetate obtained in Example 2were packed in a stainless steel column having a length of 25 cm and aninner diameter of 0.46 cm by slurry process. The high performance liquidchromatograph used was TRIROTAR-SR (a product of Japan SpectroscopicCo., Ltd.) and the detector was UVIDEC-V. The results of the resolutionof trans-stilbene oxide are shown in Table 2. The resolution factor α(1.51) obtained in this application example was far higher than that(1.22) obtained with cellulose triacetate in the same manner as above.

                  TABLE 2                                                         ______________________________________                                                           Reso-                                                                 capacity                                                                              lution  Rate of   Flow                                                ratio   factor  separation                                                                              rate                                     racemates    k.sub.1 '                                                                            k.sub.2 '                                                                            α                                                                             Rs      ml/min                               ______________________________________                                         ##STR3##    1.08   1.64   1.51  0.6     1.0                                  ______________________________________                                         Solvent: hexane/2propanol (9:1)                                          

SYNTHESIS EXAMPLE 4 Synthesis of Cellulose Tripropionate

3.0 g of the cellulose prepared in Synthesis Example 2 was suspended in30 ml of dry pyridine. 20 ml of propionic anhydride was added to thesuspension and the reaction was carried out at 100° C. The reactionmixture was added to 200 ml of hexane to form a precipitate, which wasfiltered and dried to obtain 4.28 g of a product. The product wasreesterified by heating to 100° C. together with 30 ml of dry pyridineand 10 ml of propionic anhydride for 6 h. The product was precipitatedfrom 200 ml of ether and petroleum benzene at 1:1. The precipitate wasfiltered, washed with 2-propanol and then methanol and dried.

The product had a limiting viscosity number in dichloromethane/methanol(9:1) of 0.345 (25° C.). In the I.R. spectrum, no ν_(OH) was observed.This suggested that the product was a triester.

EXAMPLE 3

1.2 g of cellulose tripropionate obtained in Synthesis Example 4 wasdissolved in 7.5 ml of dichloromethane. The solution was thoroughlymixed with 3.6 g of the silica beads obtained in Synthesis Example 1.The solvent was distilled off under reduced pressure to obtain apowdery, supported material.

APPLICATION EXAMPLE 3

The optical resolution of compounds was conducted in the same manner asin Application Example 1 except that the silica beads carrying cellulosetripropionate obtained in Example 3 were used. The results are shown inTable 3.

                  TABLE 3                                                         ______________________________________                                                                   Resolution                                                        capacity ratio                                                                            factor                                             racemates        k.sub.1 '                                                                              k.sub.2 '                                                                              α                                    ______________________________________                                         ##STR4##        1.44(+)   2.92(-) 2.03                                        ##STR5##        8.2      12.8     1.56                                       ______________________________________                                         Eluent: hexane/2propanol (9:1)                                                Flow rate: 0.5 ml/min                                                    

SYNTHESIS EXAMPLE 5 Synthesis of Chitin Diacetate

Chitin (a product of Nan'yo Kasei K.K.) was acetylated in perchloricacid by a process disclosed in literature (Norio Nishi, Junzo Noguchi,Seiichi Tokura, Hiroyuki Shiota, "Polymer Journal" 11, 27 (1979)) toobtain chitin diacetate.

EXAMPLE 4

1.2 g of chitin diacetate obtained in Synthesis Example 5 was dissolvedin a mixture of 4 ml of dichloromethane, 0.5 ml of acetic anhydride and3.5 ml of trifluoroacetic acid. The solution was left to stand at roomtemperature (about 25° C.) for 2 days to reduce its viscosity. Thesolution was filtered through a glass filter (G3) and mixed well with3.2 g of the silica beads obtained in Synthesis Example 1. The solventwas distilled off under reduced pressure to obtain a powdery, supportedmaterial.

APPLICATION EXAMPLE 4

The optical resolution of compounds was conducted in the same manner asin Application Example 1 except that the silica beads carrying chitindiacetate obtained in Example 4 were used. The results are shown inTable 4.

                  TABLE 4                                                         ______________________________________                                                                   Resolution                                                        capacity ratio                                                                            factor                                             racemates        k.sub.1 '                                                                              k.sub.2 '                                                                              α                                    ______________________________________                                         ##STR6##         2.43(+)  2.98(-) 1.22                                        ##STR7##        14.4(-)  16.5(+)  1.15                                       ______________________________________                                         Eluent: hexane/2propanol (9:1)                                                Flow rate: 0.5 ml/min                                                    

SYNTHESIS EXAMPLE 6 Synthesis of Amylose Triacetate

2 g of Amylose DEX-III (a product of Hayashibara added to 10 ml ofwater. 2.2 ml of 30% sodium hydroxide was added thereto under coolingwith ice to obtain a solution. Then, sodium hydroxide was neutralizedwith acetic acid. This amylose solution was added to ethanol to form aprecipitate, which was washed thoroughly with ethanol and the ethanolwas replaced with ether. 50 ml of dehydrated pyridine was added theretoand the mixture was filtered. This treatment was repeated three times.70 ml of pyridine and 20 ml of acetic anhydride were added to theamylose wetted with pyridine and the reaction was carried out at 100° C.for 6.5 h.

After completion of the reaction, the reaction mixture was added toethanol to form a precipitate, which was collected and washed completelywith ethanol, dried in vacuo and dissolved in methylene chloride. Thesolution was filtered through a G3 glass filter under pressure. Thefiltrate was added to ethanol to form a precipitate, which was washedwith ethanol and dried.

The product was dissolved in methylene chloride and the solution wasapplied to a rock salt plate and dried to obtain a sample. In the I.R.spectrum of the sample, no absorption due to unsubstituted alcoholgroups of the cellulose was observed.

trans-stilbene oxide: α=1.14 (+).

EXAMPLE 5

1.2 g of amylose triacetate obtained in Synthesis Example 6 wasdissolved in 7.5 ml of dichloromethane. The solution was filteredthrough a G3 glass filter and then mixed well with 3.5 g of the silicabeads obtained in Synthesis Example 1. The solvent was distilled offunder reduced pressure to obtain a powdery, supported material.

APPLICATION EXAMPLE 5

The optical resolution of trans-stilbene oxide was conducted in the samemanner as in Application Example 1 except that the silica beads treatedin Example 5 were used. The resolution factor (α) obtained was 1.14.

Eluent: hexane/2-propanol (9:1)

Flow rate: 0.5 ml/min.

SYNTHESIS EXAMPLE 7

10 g of silica beads (LiChrospher SI 1000; a product of Merck & Co.) wasplaced in a 200 ml round-bottom flask with a side arm. Aftervacuum-drying in an oil bath at 120° C. for 3 h, N₂ was introducedtherein. 100 ml of toluene which had been preliminarily distilled in thepresence of CaH₂ was added to the silica beads. 3 ml ofdiphenyldimethoxysilane (KBM 202; a product of Shin'etsu Kagaku Co.,Ltd.) was added to the mixture and they were stirred together and thenreacted at 120° C. for 1 h. After distilling off 3 to 5 ml of toluene,the reaction was carried out at 120° C. for 2 h. The mixture wasfiltered through a glass filter, washed with 50 ml of toluene threetimes and then with 50 ml of methanol three times and dried in vacuum at40° C. for 1 h.

About 10 g of the silica beads were placed in the 200 ml round-bottomflask with a side arm. After vacuum drying at 100° C. for 3 h, thepressure was returned to the atmospheric pressure and the mixture wascooled to room temperature. Then N₂ was introduced therein. 100 ml ofdistilled toluene was added to the dried silica beads. 1 ml ofN,O-bis(trimethylsilyl)acetamide (a trimethylsilylating agent) was addedthereto and the mixture was stirred to effect the reaction at 115° C.for 3 h. The reaction mixture was filtered through a glass filter,washed with toluene and dried under vacuum for about 4 h.

SYNTHESIS EXAMPLE 8 Synthesis of β-1,4-Mannan Tribenzoate

Albumens of seeds of ivory palm were treated by a process disclosed inliterature [see G. O. Aspinall et al., "J. Chem. Soc.", 3184 (1953)] toobtain mannan B from a high molecular weight fraction. 1.5 g of powderof mannan B was mixed with 70 ml of dehydrated pyridine, 7.7 ml ofdehydrated triethylamine and 50 mg of 4-dimethylaminopyridine. 10.7 mlof benzoyl chloride was added thereto under stirring. The reaction wascarried out at 100° C. for 5 h. After cooling, the product was added to400 ml of ethanol under stirring to form a precipitate, which wasfiltered through a glass filter and washed thoroughly with ethanol.After vacuum drying, the product was dissolved in 30 ml of methylenechloride and an insoluble matter was removed. The product wasreprecipitated from 400 ml of ethanol. The precipitate was collected byfiltration, washed with ethanol, dehydrated and dried.

The product was dissolved in methylene chloride. The solution wasapplied to a common salt tablet and dried. The infrared absorptionspectrum of the product had the following characteristic absorptionbands:

3070 cm⁻¹ : stretching vibration of aromatic C--H,

1730 cm⁻¹ : stretching vibration of C═O of carboxylic acid ester,

1605, 1495, 1455 cm⁻¹ : skeletal vibration due to stretching of carbonand carbon in the benzene ring,

1270 cm⁻¹ : stretching vibration of C--O of ester,

1030 to 1200 cm⁻¹ : stretching vibration of C--O--C of mannan, and

690 to 900 cm⁻¹ : out-of-plane deformation vibration of benzene ring.

Substantially no absorption at around 3450 cm⁻¹ due to OH of mannan wasobserved. This fact suggested that the product substantially comprised atrisubstituted compound. In the proton NMR spectrum determined in CDCl₃,the characteristic absorptions were as follows:

6.8 to 8.4 ppm: proton of benzene ring,

2.8 to

6.0 ppm: protons of mannan ring and methylene in position 6.

The ratio of these absorption intensities was 15:7.

SYNTHESIS EXAMPLE 9 Synthesis of Tribenzoyl-β-1,4-Chitosan

10 g of purified chitosan was dissolved in 1000 ml of water containing40 ml of conc. hydrochloric acid. The solution was kept at 73° C. for 5h. The solution was concentrated by means of a rotary evaporator andthen neutralized with 27 ml of aqueous ammonia (28%) to precipitatechitosan. The precipitate was collected by filtration, washed withwater, ethanol and then ether each twice and dried in vacuo. Yield: 9.71g.

1.0 g of obtained chitosan was dissolved in 30 ml of water containing0.5 ml of conc. hydrochloric acid. The solution was freeze-dried. 30 mlof pyridine, 0.05 g of 4-dimethylaminopyridine, 8 ml of triethylamineand 10 ml of benzoyl chloride were added to the residue and the mixturewas kept at 100° to 105° C. under stirring for 7 h. The resultingsuspension was added to ethanol to form a precipitate, which wasfiltered, washed with ether and then with dichloromethane and dried invacuo. Yield: 3.0 g. In the I.R. spectrum, two carbonyl stretchingvibrations were observed at 1720 and 1660 cm⁻¹. This fact suggested thatthe product was tribenzoylchitosan.

EXAMPLE 6

1.2 g of β-1,4-mannan tribenzoate obtained in Synthesis Example 8 wasdissolved in a mixture of 12.5 ml of dichloromethane and 3.5 ml ofacetone. 6.4 g of the silica gel particles obtained in Synthesis Example7 were impregnated with the solution. The solvent was distilled offunder reduced pressure to obtain a powdery, supported material.

EXAMPLE 7

1.2 g of tribenzoylchitosan obtained in Synthesis Example 9 wasdissolved in a mixture of 5 ml of dichloromethane and 4.5 ml ofdichloroacetic acid. The solution was filtered. 3.2 g of the silica gelobtained in Synthesis Example 1 was impregnated with 7.5 ml of thesolution. The vessel was heated with hot water under reduced pressurerealized by means of a vacuum pump to remove the solvent. A powdery,supported material was thus obtained.

APPLICATION EXAMPLE 6

The silica beads carrying mannan tribenzoate obtained in Example 6 werepacked in a stainless steel column having a length of 25 cm and an innerdiameter of 0.46 cm by slurry process. The high performance liquidchromatograph used was TRIROTAR-SR (a product of Japan SpectroscopicCo., Ltd.) and the detector was UVIDEC-V. The results of the resolutionof 2-phenylcyclohexanone are shown in Table 5.

                  TABLE 5                                                         ______________________________________                                                             Reso-   Rate of                                                       capacity                                                                              lution  separa- Flow                                                  ratio   factor  tion    rate                                     racemates      k.sub.1 '                                                                            k.sub.2 '                                                                            α                                                                             Rs    ml/min                               ______________________________________                                         ##STR8##      3.80   4.02   1.06  0.5   0.5                                  ______________________________________                                         Solvent: hexane/2propanol (9:1)                                          

APPLICATION EXAMPLE 7

The silica beads carrying tribenzoylchitosan obtained in Example 7 weresuspended in methanol. The suspension was packed in a stainless steelcolumn having a length of 25 cm and an inner diameter of 0.46 cm byslurry process. The high performance liquid chromatograph used wasTRIROTAR-SR (a product of Japan Spectroscopic Co., Ltd.) and thepolarimeter detector was DIP-181 (a product of Japan Spectroscopic Co.,Ltd.). The results of the resolution of racemic compounds are shown inTable 6.

In the determination effected by using the polarimeter as the detectorof the high performance chromatograph, the terms were defined asfollows: ##EQU2##

                  TABLE 6                                                         ______________________________________                                                                           Flow rate                                  racemates     l.sub.1 '                                                                            l.sub.2 '                                                                              β                                                                             (ml/min)                                   ______________________________________                                                      1.56   1.88     1.21 1.0                                        ______________________________________                                         Solent: hexane/2propanol (9:1)                                           

SYNTHESIS EXAMPLE 10 Synthesis of β-1,4-Xylan Benzoate

5.0 g of xylan (a product of Tokyo Kasei Co.) was dispersed in 10 ml ofwater. A 30% aqueous sodium hydroxide solution was added to thedispersion under cooling with ice until xylan had been dissolved to forma transparent solution. The solution was added to 150 ml of methanolcontaining 3 ml of acetic acid to form a precipitate, which was filteredand washed with methanol. About a half of the precipitate was suspendedin a mixture of 40 ml of benzene and 40 ml of pyridine. Benzene wasdistilled off from the reaction system through a 20 cm column. 20 ml ofbenzoyl chloride was added thereto and the mixture was kept at 90° C.for 10 h. The mixture was cooled and then added to 500 ml of ethanol toform a precipitate, which was filtered, washed and dried. The productwas dissolved in dichloromethane. An insoluble matter was removed byfiltration. Dichloromethane was distilled off under reduced pressure toobtain xylan dibenzoate. In the I.R. absorption spectrum of thisproduct, the absorption due to the stretching vibration of OH was weak.

EXAMPLE 8

1.2 g of β-1,4-xylan dibenzoate obtained in Synthesis Example 10 wasdissolved in 7.5 ml of dichloromethane. The solution was filteredthrough a glass filter (G2) and the filtrate was mixed with 3.4 g of thesilica beads obtained in Synthesis Example 7. The solvent was distilledoff under reduced pressure to obtain a powdery, supported material.

APPLICATION EXAMPLE 8

The results of the optical resolution of racemic compounds conductedwith the supported material obtained in Example 8 under the sameconditions as in Application Example 6 are shown in Table 7.

                  TABLE 7                                                         ______________________________________                                                                  Resolution                                                       capacity ratio                                                                             factor                                              racemates      k.sub.1 ' k.sub.2 '                                                                              α                                     ______________________________________                                         ##STR9##      1.75(+)   2.04(-)  1.17                                         ##STR10##     4.91(-)   5.74(+)  1.17                                        ______________________________________                                         Eluent: hexane/2propanol (9:1)                                                Flow rate: 0.5 ml/min                                                    

SYNTHESIS EXAMPLE 11

20 g of purified chitosan (a product of Kyowa Kasei Co.) was dispersedin 1 l of water. 40 ml of conc. hydrochloric acid was added slowly tothe dispersion to dissolve chitosan. The resulting solution was kept at80° C. for 5 h and then cooled. The solids suspended therein wasfiltered out. The filtrate was concentrated to a volume of 200 ml bymeans of a rotary evaporator. The liquid was made alkaline with excessaqueous ammonia. Chitosan having a reduced molecular weight was thusprecipitated. After collection, the product was washed with water andthen ethanol and dried. 1.0 g of the resulting chitosan was dispersed in10 ml of water. Hydrochloric acid was added in portions to thedispersion until a solution was obtained. The solution was added to 60ml of methanol containing 3 ml of aqueous ammonia (28%) to precipitatechitosan. After filtration, the product was washed with methanol twiceand then with ether twice. Chitosan containing the solvent was added to60 ml of pyridine. A part (about 30 ml) of pyridine was distilled off.10 ml of benzoyl chloride was added dropwise to the obtainedchitosan-containing pyridine and the mixture was kept at 80 ° to 90° C.for 8 h. A suspension thus formed was added to cooled methanol to form aprecipitate. After filtration, the product was washed with methanol,acetone and dichloromethane and then dried.

EXAMPLE 9

1.05 g of tribenzoylchitosan obtained in Synthesis Example 11 wasdissolved in a mixture of 4.0 ml of dichloromethane, 3.0 ml oftrifluoroacetic acid and 0.5 ml of benzoyl chloride. The solution wasmixed with 3.2 g of the silica beads obtained in Synthesis Example 7.The solvent was distilled off under reduced pressure to obtain apowdery, supported material.

APPLICATION EXAMPLE 9

The optical resolution of racemic compounds was conducted with thesupported material obtained in Example 9 in the same manner as inApplication Example 6. The results are shown in Table 8. It was notedthat Troger's bases which could not be resolved with cellulosetribenzoate were resolved.

                                      TABLE 8                                     __________________________________________________________________________                       capacity ratio                                                                          Resolution factor                                racemates          k.sub.1 '                                                                          k.sub.2 '                                                                          α                                          __________________________________________________________________________     ##STR11##          1.71(-)                                                                            1.85(+)                                                                           1.08                                              ##STR12##          1.31(+)                                                                            1.61(-)                                                                           1.23                                              ##STR13##          3.93(-)                                                                            4.28(+)                                                                           1.09                                              ##STR14##          6.64                                                                               7.28                                                                              1.10                                              ##STR15##         14.6(+)                                                                            15.9(-)                                                                            1.09                                             __________________________________________________________________________     Eluent: hexane/2propanol (9:1)                                                Flow rate: 0.5 ml/min.                                                   

SYNTHESIS EXAMPLE 12 Synthesis of Mannan A Tribenzoate

Albumens of seeds of ivory palm were treated by a process disclosed inliterature [see G. O. Aspinall et al., "J. Chem. Soc.", 3184 (1953)] toobtain mannan A from a low molecular weight fraction.

1.5 g of mannan A was dispersed in a solution comprising 70 ml ofpyridine, 7.7 ml of triethylamine and 50 mg of 4-dimethylaminopyridine.10.7 ml of benzoyl chloride was added to the dispersion and the reactionwas carried out at 100° C. for 5 h. After cooling, the reaction mixturewas added to ethanol to form a precipitate, which was washed thoroughlywith ethanol and dried. The dried sample was dissolved in methylenechloride. The solution was filtered through a glass filter (G3). Thefiltrate was added to ethanol to form a precipitate, which was washedthoroughly with ethanol and dried.

EXAMPLE 10

1.2 g of mannan A tribenzoate obtained in Synthesis Example 12 wasdissolved in 7.5 ml of dichloromethane. The solution was filteredthrough a glass filter (G-2). The filtrate was mixed thoroughly with 3.4g of the silica beads obtained in Synthesis Example 7. The solvent wasdistilled off under reduced pressure to obtain a powdery, supportedmaterial.

APPLICATION EXAMPLE 10

The optical resolution of racemic compounds of γ-phenyl-γ-butyrolactonewas conducted with the supported material obtained in Example 10 in thesame manner as in Application Example 6. The resolution factor (α) was1.22 [(+) compound being eluted first]. The eluent was hexane/2-propanol(9:1). The flow rate was 0.5 ml/min.

SYNTHESIS EXAMPLE 13 Synthesis of Dextran Tribenzoate

3.0 g of dextran (a product of Nakai Kagaku Yakuhin Co. having amolecular weight of 50,000 to 70,000) was dissolved in 10 ml of water.Pyridine was added to the solution to precipitate dextran. Pyridine wasremoved by decantation. Additional 20 ml of pyridine was added and thenremoved by the decantation. The addition and decantation of pyridine wasrepeated three times. Then, 30 ml of pyridine and 20 ml of benzoylchloride were added thereto and the mixture was kept at 70° C. for 10 h.After cooling, pyridinium chloride and unreacted dextran were removed bymeans of a glass filter (G-2). The filtrate was concentrated underreduced pressure. Methanol was added thereto to precipitate dextrantribenzoate, which was filtered, washed with methanol and dried. In theI.R. absorption spectrum of the product, the absorption due tostretching vibration of OH was weak. This fact suggested that theproduct was a trisubstituted compound.

EXAMPLE 11

1.2 g of dextran tribenzoate obtained in Synthesis Example 13 wasdissolved in 7.5 ml of dichloromethane. The solution was mixedthoroughly with 3.6 g of the silica gel obtained in Synthesis Example 7.The solvent was distilled off under reduced pressure to obtain apowdery, supported material.

APPLICATION EXAMPLE 11

The optical resolution of trans-stilbene oxide was conducted with thesupported material obtained in Example 11 in the same manner as inApplication Example 6. The resolution factor was 1.18. The enantiomerhaving (-) optical rotation was eluted first. The eluent washexane/2-propanol (9:1). The flow rate was 0.5 ml/min.

The embodiments of the invention in which an exclusive property orprivilege is claimed are defined as follows:
 1. In a method forchromatographically separating an optical isomer from a racemic mixturethereof, wherein the mixture is contacted with a separation agent underchromatographic separation conditions, the improvement which comprises:employing as the separation agent cellulose triscyclopropanecarboxylate.2. A method as claimed in claim 1, in which said mixture is conductedthrough a chromatographic column or layer to effect separation andresolution of the components of said mixture.
 3. A method as claimed inclaim 1, in which 30 percent or more of the hydroxyl groups of saidcellulose are esterified with said triscyclopropanecarboxylic acid.
 4. Amethod as claimed in claim 1, in which 85 percent or more of thehydroxyl groups of said cellulose are esterified with saidtriscyclopropanecarboxylic acid.
 5. A method for chromatographicallyseparating an optical isomer from a racemic mixture thereof whichcomprises contacting said mixture, under liquid chromatographicseparation conditions, with a chromatographic separation materialconsisting essentially of inorganic carrier particles having a particlesize of from 1 μm to 10 mm and coated with from 1 to 100% by weight ofresolving agent, based on the weight of said carrier particles, saidresolving agent consisting essentially of cellulosetriscyclopropanecarboxylate.
 6. A method as claimed in claim 5 in whichsaid inorganic carrier is porous and has an average pore diameter offrom 10 Å to 100 μm.
 7. A method as claimed in claim 5 in which saidinorganic carrier is selected from the group consisting of silica,alumina, magnesia, titanium oxide, glass and kaolin.
 8. A method asclaimed in claim 5 in which said inorganic carrier particles are silicabeads which have been surface treated with an organosilane compound toimprove their affinity for the resolving agent.
 9. A method as claimedin claim 8 in which said resolving agent consists of cellulosetriscyclopropanecarboxylate.